Project Summary Alzheimer?s disease (AD) is a debilitating neurodegenerative disease that is growing in prevalence despite decades of investment in the research and development of diagnostics and therapeutics. Because of the high healthcare burden and devastating effects of AD on cognitive function and quality of life, early biomarkers of disease and interventions to prevent and treat AD are a public health priority. The search for biomarkers and treatments has turned to glycobiology, the study of complex sugars attached to proteins and lipids. The post- translational glycosylation of proteins and lipids plays a role in a number of critical biological processes including cell-to-cell communication and signaling, and protein structure and function. Recently, major alterations in glycosylation have been documented in the brains, cerebrospinal fluid, and blood of AD patients even before the onset of advanced cognitive decline. Glycosylation is likely to be causally involved in AD, thus measuring glycosylation in blood is a promising strategy for discovering early biomarkers. Apolipoprotein E (ApoE) is the single greatest genetic risk factor for AD. Carriers of the ApoE4 isoform are 4-12 times more likely to develop AD depending on the number of copies of ApoE4, and develop the disease as many as 20 years earlier. ApoE is a glycosylated protein, and nearly all of the proteins, carriers, and receptors involved in its metabolism are also glycosylated. The structure of ApoE heavily influences its functionality. Glycosylation is known to alter protein structure yet neither the glycosylation of ApoE, nor the overall glycosylation in the brain and in the blood have been adequately characterized in Alzheimer?s disease patients because the lack of precise analytical tools for measuring glycosylation has been a technological barrier to progress. Our group has pioneered the development of advanced and sensitive liquid chromatography-mass spectrometry methods as tools to precisely measure glycosylation alterations across hundreds of lipids and proteins simultaneously. Our method is rapid-throughput and can monitor the site-specificity, structure specificity, and linkage specificity of all attached glycans in hundreds of clinical samples with high reproducibility, accuracy and sensitivity. The three specific aims of this project are to map the global glycosylation alterations in the brains and blood of Alzheimer?s disease patients compared with controls, to characterize the site-specific changes in glycosylation of ApoE, and to document the effects of glycosylation changes on critical pathways involved in Alzheimer?s disease pathology. Importantly, the differences in glycosylation in AD will be mapped within each ApoE genotype, which will enable the discovery of precision medicine based solutions. Successful completion of this project will lead to the development of new glycosylation-based biomarkers for the early detection of Alzheimer?s disease and ApoE-specific targets for the development of novel therapeutics in AD.